Glycogen phosphorylase from streptococcus pneumoniae

ABSTRACT

The invention provides glycogen polypeptides, fusion polypeptides and compositions there comprising. Also provided are preferred methods for utilizing these polypeptides as diagnostic reagents and in diagnostic assays to screen for microbial infections in organisms and materials.

This is a divisional of application Ser. No. 08/896,590, filed Jul. 17,1997 now U.S. Pat. No. 5,882,885.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides andpolypeptides, and their production and uses, as well as their variants,agonists and antagonists, and their uses. In particular, in these and inother regards, the invention relates to novel polynucleotides andpolypeptides of the glycogen phosphorylase family, hereinafter referredto as “glycogen phosphorylase”.

BACKGROUND OF THE INVENTION

The Streptococci make up a medically important genera of microbes knownto cause several types of disease in humans, including, for example,otitis media, conjunctivitis, pneumonia, bacteremia, meningitis,sinusitis, pleural empyema and endocarditis, and most particularlymeningitis, such as for example infection of cerebrospinal fluid. Sinceits isolation more than 100 years ago, Streptococcus pneumoniae has beenone of the more intensively studied microbes. For example, much of ourearly understanding that DNA is, in fact, the genetic material waspredicated on the work of Griffith and of Avery, Macleod and McCartyusing this microbe. Despite the vast amount of research with S.pneumoniae, many questions concerning the virulence of this microberemain. It is particularly preferred to employ Streptococcal genes andgene products as targets for the development of antibiotics.

The frequency of Streptococcus pneumoniae infections has risendramatically in the past 20 years. This has been attributed to theemergence of multiply antibiotic resistant strains and an increasingpopulation of people with weakened immune systems. It is no longeruncommon to isolate Streptococcus pneumoniae strains which are resistantto some or all of the standard antibiotics. This has created a demandfor both new anti-microbial agents and diagnostic tests for thisorganism.

Glycogen phosphorylase is a catabolic gene involved in the breakdown ofglycogen, an energy storage carbohydrate in many organisms. It has beendetected in a wide range of bacteria (Romeo T, Kumar A, Preiss J GeneOct. 30, 1988; 70(2):363-376, Kiel J A, Boels J M, Beldman G, Venema GMol Microbiol Jan. 11, 1994(l):203-218 and Preiss J, Romeo T Adv MicrobPhysiol 1989, 30:183-238) The breakdown of glycogen may be particularlyimportant in adverse situations for bacterial growth such as infectionin mammals.

Clearly, there is a need for factors, such as the novel compounds of theinvention, that have a present benefit of being useful to screencompounds for antibiotic activity. Such factors are also useful todetermine their role in pathogenesis of infection, dysfunction anddisease. There is also a need for identification and characterization ofsuch factors and their antagonists and agonists which can play a role inpreventing, ameliorating or correcting infections, dysfunctions ordiseases.

The polypeptides of the invention have amino acid sequence homology to aknown maltodextrin phosphorylase (phsm_ecoli) protein.

SUMMARY OF THE INVENTION

It is an object of the invention to provide polypeptides that have beenidentified as novel glycogen phosphorylase polypeptides by homologybetween the amino acid sequence set out in Table 1 [SEQ ID NO: 2] and aknown amino acid sequence or sequences of other proteins such asMALTODEXTRIN PHOSPHORYLASE (PHSM_ECOLI) protein.

It is a further object of the invention to provide polynucleotides thatencode glycogen phosphorylase polypeptides, particularly polynucleotidesthat encode the polypeptide herein designated glycogen phosphorylase.

In a particularly preferred embodiment of the invention thepolynucleotide comprises a region encoding glycogen phosphorylasepolypeptides comprising the sequence set out in Table 1 [SEQ ID NO:1]which includes a full length gene, or a variant thereof.

In another particularly preferred embodiment of the invention there is anovel glycogen phosphorylase protein from Streptococcus pneumoniaecomprising the amino acid sequence of Table 1 [SEQ ID NO:2], or avariant thereof.

In accordance with another aspect of the invention there is provided anisolated nucleic acid molecule encoding a mature polypeptide expressibleby the Streptococcus pneumoniae 0100993 strain contained in thedeposited strain.

A further aspect of the invention there are provided isolated nucleicacid molecules encoding glycogen phosphorylase, particularlyStreptococcus pneumoniae glycogen phosphorylase, including mRNAs, cDNAs,genomic DNAs. Further embodiments of the invention include biologically,diagnostically, prophylactically, clinically or therapeutically usefulvariants thereof, and compositions comprising the same.

In accordance with another aspect of the invention, there is providedthe use of a polynucleotide of the invention for therapeutic orprophylactic purposes, in particular genetic immunization. Among theparticularly preferred embodiments of the invention are naturallyoccurring allelic variants of glycogen phosphorylase and polypeptidesencoded thereby.

Another aspect of the invention there are provided novel polypeptides ofStreptococcus pneumoniae referred to herein as glycogen phosphorylase aswell as biologically, diagnostically, prophylactically, clinically ortherapeutically useful variants thereof, and compositions comprising thesame.

Among the particularly preferred embodiments of the invention arevariants of glycogen phosphorylase polypeptide encoded by naturallyoccurring alleles of the glycogen, phosphorylase genie.

In a preferred embodiment of the invention there are provided methodsfor producing the aforementioned glycogen phosphorylase polypeptides.

In accordance with yet another aspect of the invention, there areprovided inhibitors to such polypeptides, useful as antibacterialagents, including, for example, antibodies.

In accordance with certain preferred embodiments of the invention, thereare provided products, compositions and methods for assessing glycogenphosphorylase expression, treating disease, for example, otitis mediaconjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, assaying genetic variation,and administering a glycogen phosphorylase polypeptide or polynucleotideto an organism to raise an immunological response against a bacteria,especially a Streptococcus pneumoniae bacteria.

In accordance with certain preferred embodiments of this and otheraspects of the invention there are provided polynucleotides thathybridize to glycogen phosphorylase polynucleotide sequences,particularly under stringent conditions.

In certain preferred embodiments of the invention there are providedantibodies against glycogen phosphorylase polypeptides.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a polypeptide or polynucleotide ofthe invention comprising: contacting a polypeptide or polynucleotide ofthe invention with a compound to be screened under conditions to permitbinding to or other interaction between the compound and the polypeptideor polynucleotide to assess the binding to or other interaction with thecompound, such binding or interaction being associated with a secondcomponent capable of providing a detectable signal in response to thebinding or interaction of the polypeptide or polynucleotide with thecompound; and determining whether the compound binds to or otherwiseinteracts with and activates or inhibits an activity of the polypeptideor polynucleotide by detecting the presence or absence of a signalgenerated from the binding or interaction of the compound with thepolypeptide or polynucleotide.

In accordance with yet another aspect of the invention, there areprovided glycogen phosphorylase agonists and antagonists, preferablybacteriostatic or bactericidal agonists and antagonists.

In a further aspect of the invention there are provided compositionscomprising a glycogen phosphorylase polynucleotide or a glycogenphosphorylase polypeptide for administration to a cell or to amulticellular organist

Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following descriptions and from reading the otherparts of the present disclosure.

Glossary

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein.

“Host cell” is a cell which has been transformed or transfected, or iscapable of transformation or transfection by an exogenous polynucleotidesequence.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as the case may be, as determined by the match betweenstrings of such sequences. “Identity” and “similarity” can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data,Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math, 48: 1073(1988). Preferred methods to determine identity are designed to give thelargest match between the sequences tested. Methods to determineidentity and similarity are codified in publicly available computerprograms. Preferred computer program methods to determine identity andsimilarity between two sequences include, but are not limited to, theGCG program package (Devereux, J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol 215: 403-410 (1990). The BLAST X program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIHBethesda, Md. 20894; Altschul, S., et al, J. Mol. Biol. 215: 403410(1990). As an illustration, by a polynucleotide having a nucleotidesequence having at least, for example, 95% “identity” to a referencenucleotide sequence of SEQ ID NO: 1 it is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations per each 100 nucleotides of the reference nucleotide sequenceof SEQ ID NO: 1. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence. Analogously, by a polypeptide having an amino acidsequence having at least, for example, 95% identity to a reference aminoacid sequence of SEQ ID NO:2 is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of SEQ ID NO: 2. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence. These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

“Isolated” means altered “by the hand of man” from its natural state,i.e., if it occurs in nature, it has been changed or removed from itsoriginal environment, or both. For example, a polynucleotide or apolypeptide naturally present in a living organism is not “isolated,”but the same polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein.

“Polynucleotide(s)” generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotide(s)” include, without limitation, single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded regions, or a mixture of single- and double-strandedregions. In addition, “polynucleotide” as used herein refers totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.As used herein, the term “polynucleotide(s)” also includes DNAs or RNAsas described above that contain one or more modified bases. Thus, DNAsor RNAs with backbones modified for stability or for other reasons are“polynucleotide(s)” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. It will be appreciated that a great variety ofmodifications have been made to DNA and RNA that serve many usefulpurposes known to those of skill in the art The term “polynucleotide(s)”as it is employed herein embraces such chemically, enzymatically ormetabolically modified forms of polynucleotides, as well as the chemicalforms of DNA and RNA characteristic of viruses and cells, including, forexample, simple and complex cells. “Polynucleotide(s)” also embracesshort polynucleotides often referred to as oligonucleotide(s).

“Polypeptide(s)” refers to any peptide or protein comprising two or moreamino acids joined to each other by peptide bonds or modified peptidebonds. “Polypeptide(s)” refers to both short chains, commonly referredto as peptides, oligopeptides and oligomers and to longer chainsgenerally referred to as proteins. Polypeptides may contain amino acidsother than the 20 gene encoded amino acids. “Polypeptide(s)” includethose modified either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques. Such modifications are well described in basic texts and inmore detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Itwill be appreciated that the same type of modification may be present inthe same or varying degree at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains, and the amino or carboxyl termini.Modifications include, for example, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-liking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formulation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins, such as arginylation, and ubiquitination. See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993) and Wold, F.,Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). Polypeptides may be branched or cyclic, with or withoutbranching. Cyclic, branched and branched circular polypeptides mayresult from post-translational natural processes and may be made byentirely synthetic methods, as well.

“Variant(s)” as the tern is used herein, is a polynucleotide orpolypeptide that differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. Nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide encoded by the reference sequence, as discussed below. Atypical variant of a polypeptide differs in amino acid sequence fromanother, reference polypeptide. Generally, differences are limited sothat the sequences of the reference polypeptide and the variant areclosely similar overall and, in many regions, identical. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A substituted orinserted amino acid residue may or may not be one encoded by the geneticcode. A variant of a polynucleotide or polypeptide may be a naturallyoccurring such as an allelic variant, or it may be a variant that is notknown to occur naturally. Non-naturally occurring variants ofpolynuclotides and polypeptides may be made by mutagenesis techniques,by direct synthesis, and by other recombinant methods known to skilledartisans.

DESCRIPTION OF THE INVENTION

The invention relates to novel glycogen phosphorylase polypeptides andpolynucleotides as described in greater detail below. In particular, theinvention relates to polypeptides and polynucleotides of a novelglycogen phosphorylase of Streptococcus pneumoniae, which is related byamino acid sequence homology to E. coli maltodextrin phosphorylase(PHSM_ECOLI) polypeptide. The invention relates especially to glycogenphosphorylase having the nucleotide and amino acid sequences set out inTable 1 [SEQ ID NO: 1] and Table 1 [SEQ ID NO: 2] respectively, and tothe glycogen phosphorylase nucleotide sequences of the DNA in thedeposited strain and amino acid sequences encoded thereby.

TABLE 1 glycogen phosphorylase Polynucleotide and Polypeptide Sequences(A) Sequences from Streptococcus pneumoniae glycogen phosphorylasepolynucleotide sequence [SEQ ID NO:1]. 5′-1 ATGTTATCAC TACAAGAATTTGTACAAAAT CGTTACAATA AAACCATTGC 51 AGAATGTAGC AATGAAGAGC TTTACCTTGCTCTTCTTAAC TACAGCAAGC 101 TTGCAAGCAG CCAAAAACCA GTCAACACTG GTAAGAAAAAAGTTTACTAC 151 ATCTCAGCTG AGTTCTTGAT TGGTAAACTC TTGTCAAACA ACTTGATTAA201 CCTTGGTCTT TACGACGATG TTAAAAAAGA ACTTGCAGCT GCAGGTAAAG 251ACTTGATCGA AGTTGAAGAA GTTGAATTGG AACCATCTCT TGGTAATGGT 301 GGTTTGGGACGTTTGGCTGC CTGCTTTATC GACTCAATTG CTACTCTTGG 351 TTTGAATGGT GACGGTGTTGGTCTTAACTA CCACTTTGGT CTTTTCCAAC 401 AAGTTCTTAA AAACAACCAA CAAGAAACAATTCCAAATGC ATGGTTGACA 451 GAGCAAAACT GGTTGGTTCG CTCAAGCCGT AGCTACCAAGTACCATTTGC 501 AGACTTTACT TTGACATCAA CTCTTTACGA TATTGATGTT ACTGGTTATG551 AAACAGCGAC TAAAAACCGC TTGCGTTTGT TTGACTTGGA TTCAGTTGAT 601TCTTCTATTA TTAAAGATGG TATCAACTTT GACAAGACAG ATATCGCTCG 651 CAACTTGACTCTCTTCCTTT ACCCAGATGA TAGTGACCGT CAAGGTGAAT 701 TGCTCCGTAT CTTCCAACAATACTTCATGG TTTCAAACGG TGCGCAATTG 751 ATCATCGACG AAGCAATCGA AAAAGGAAGCAACTTGCATG ACCTTGCTGA 801 CTACGCAGTT GTCCAAATCA ACGATACTCA CCCATCAATGGTGATTCCTG 851 AATTGATTCG TCTTTTGACT GCACGTGGTA TCGAGCTTGA CGAAGCAATC901 TCAATTGTTC GTAGCATGAC TGCCTACACT AACCACACAA TCCTTGCTGA 951GGCGCTTGAA AAATGGCCTC TTGAATTCTT GCAAGAAGTG GTTCCTCACT 1001 TGGTACCAATCATCGAAGAA TTGGACCGTC GTGTGAAGGC AGAGTACAAA 1051 GATCCAGCTG TTCAAATCATCGATGAGAGC GGACGTGTTC ACATGGCTCA 1101 CATGGATATC CACTACGGAT ACAGTGTTAACGGGGTTGCA GCACTTCATA 1151 CTGAAATCTT GAAAAATTCT GAGTTGAAAG CCTTCTACGACCTTTACCCA 1201 GAAAAGTTCA ACAACAAAAC AAACGGTATC ACTTTCCGTC GTTGGCTTAT1251 GCATGCTAAC CCAAGATTGT CTCACTACTT GGATGAGATT CTTGGAGATG 1301GTTGGCACCA TGAAGCAGAT GAGCTTGAAA AACTTTTGTC TTATGAAGAC 1351 AAAGCAGCTGTCAAAGAAAA ATTGGAAAGC ATCAAGGCTC ACAACAAACG 1401 TAAATTGGCT CGTCACTTGAAAGAACACCA AGGTGTGGAA ATCAATCCAA 1451 ATTCTATCTT TGATATCCAA ATCAAACGTCTTCACGAGTA CAAACGCCAA 1501 CAAATGAACG CTTTGTACGT GATCCACAAA TACCTTGACATCAAAGCTGG 1551 TAACATCCCT GCTCGTCCAA TCACAATCTT CTTTGGTGGT AAAGCAGCTC1601 CAGCCTACAC AATCGCTCAA GACATTATCC ATTTAATCCT TTGCATGTCA 1651GAAGTTATTG CTAACGATCC AGCAGTAGCT CCACACTTGC AAGTAGTTAT 1701 GGTTGAAAACTACAACGTTA CTGCAGCAAG TTTCCTTATC CCAGCATGTG 1751 ATATCTCAGA ACAAATCTCACTTGCTTCTA AAGAAGCTTC AGGTACTGGT 1801 AACATGAAAT TCATGTTGAA CGGAGCTTTGACACTTGGTA CTATGGACGG 1851 TGCTAACGTG GAAATCGCTG AGTTGGTTGG AGAAGAAAACATCTACATCT 1901 TCGGTGAAGA TTCAGAAACT GTTATCGACC TTTACGCAAA AGCAGCTTAC1951 AAATCAAGCG AATTCTACGC TCGTGAAGCT ATCAAACCAT TGGTTGACTT 2001CATCGTTAGT GATGCAGTTC TTGCAGCTGG AAACAAAGAG CGCTTGGAAC 2051 GTCTTTACAATGAATTGATC AACAAAGACT GGTTCATGAC TCTTCTTGAC 2101 TTGGAAGACT ACATCAAAGTCAAAGAGCAA ATGCTTGCTG ACTACGAAGA 2151 CCGTGACGCA TGGTTGGATA AAGTCATCGTTAACATTTCT AAAGCAGGAT 2201 TCTTCTCATC TGACCGTACA ATCGCTCAGT ATAACGAAGACATCTGGCAC 2251 TTGAACTAA -3′ (B) glycogen phosphorylase polypeptidesequence deduced from the polynucleotide sequence in this table[SEQ IDNO:2]. NH₂-1 MLSLQEFVQN RYNKTIAECS NEELYLALLN YSKLASSQKP VNTGKKKVYY 51ISAEFLIGKL LSNNLINLGL YDDVKKELAA AGKDLIEVEE VELEPSLGNG 101 GLGRLAACFIDSIATLGLNG DGVGLNYHFG LFQQVLKNNQ QETIPNAWLT 151 EQNWLVRSSR SYQVPFADFTLTSTLYDIDV TGYETATKNR LRLFDLDSVD 201 SSIIKDGINF DKTDIARNLT LFLYPDDSDRQGELLRIFQQ YFMVSNGAQL 251 IIDEAIEKGS NLHDLADYAV VQINDTHPSM VIPELIRLLTARGIELDEAI 301 SIVRSMTAYT NHTILAEALE KWPLEFLQEV VPHLVPIIEE LDRRVKKEYK351 DPAVQIIDES GRVHMAHMDI HYGYSVNGVA ALHTEILKNS ELKAFYDLYP 401EKFNNKTNGI TFRRWLMHAN PRLSHYLDEI LGDGWHHEAD ELEKLLSYED 451 KAAVKEKLESIKAANKRKLA RHLKEHQGVE INPNSIFDIQ IKRLHEYKRQ 501 QMNALYVIHK YLDIKAGNIPARPITIFFGG KAAPAYTIAQ DIIHLILCMS 551 EVIANDPAVA PHLQVVVVEN YNVTAASFLIPACDISEQIS LASKEASGTG 601 NMKFMLNGAL TLGTMDGANV EIAELVGEEN IYIFGEDSETVIDLYAKAAY 651 KSSEFYAREA IKPLVDFIVS DAVLAAGNKE RLERLYNELI NKDWFMTLLD701 LEDYIKVKEQ MLADYEDRDA WLDKVIVNIS KAGFFSSDRT IAQYNEDIWH 751 LN-COOH(C) Polynucleotide sequence embodiments[SEQ ID NO:1]. X-(R₁)_(n)-1ATGTTATCAC TACAAGAATT TGTACAAAAT CGTTACAATA AAACCATTGC 51 AGAATGTAGCAATGAAGAGC TTTACCTTGC#TCTTCTTAAC TACAGCAAGC 101 TTGCAAGCAG CCAAAAACCAGTCAACACTG GTAAGAAAAA AGTTTACTAC 151 ATCTCAGCTG AGTTCTTGAT TGGTAAACTCTTGTCAAACA ACTTGATTAA 201 CCTTGGTCTT TACGACGATG TTAAAAAAGA ACTTGCAGCTGCAGGTAAAG 251 ACTTGATCGA AGTTGAAGAA GTTGAATTGG AACCATCTCT TGGTAATGGT301 GGTTTGGGAC GTTTGGCTGC CTGCTTTATC GACTCAATTG CTACTCTTGG 351TTTGAATGGT GACGGTGTTG GTCTTAACTA CCACTTTGGT CTTTTCCAAC 401 AAGTTCTTAAAAACAACCAA CAAGAAACAA TTCCAAATGC ATGGTTGACA 451 GAGCAAAACT GGTTGGTTCGCTCAAGCCGT AGCTACCAAG TACCATTTGC 501 AGACTTTACT TTGACATCAA CTCTTTACGATATTGATGTT ACTGGTTATG 551 AAACAGCGAC TAAAAACCGC TTGCGTTTGT TTGACTTGGATTCAGTTGAT 601 TCTTCTATTA TTAAAGATGG TATCAACTTT GACAAGACAG ATATCGCTCG651 CAACTTGACT CTCTTCCTTT ACCCAGATGA TAGTGACCGT CAAGGTGAAT 701TGCTCCGTAT CTTCCAACAA TACTTCATGG TTTCAAACGG TGCGCAATTG 751 ATCATCGACGAAGCAATCGA AAAAGGAAGC AACTTGCATG ACCTTGCTGA 801 CTACGCAGTT GTCCAAATCAACGATACTCA CCCATCAATG GTGATTCCTG 851 AATTGATTCG TCTTTTGACT GCACGTGGTATCGAGCTTGA CGAAGCAATC 901 TCAATTGTTC GTAGCATGAC TGCCTACACT AACCACACAATCCTTGCTGA 951 GGCGCTTGAA AAATGGCCTC TTGAATTCTT GCAAGAAGTG GTTCCTCACT1001 TGGTACCAAT CATCGAAGAA TTGGACCGTC GTGTGAAGGC AGAGTACAAA 1051GATCCAGCTG TTCAAATCAT CGATGAGAGC GGACGTGTTC ACATGGCTCA 1101 CATGGATATCCACTACGGAT ACAGTGTTAA CGGGGTTGCA GCACTTCATA 1151 CTGAAATCTT GAAAAATTCTGAGTTGAAAG CCTTCTACGA CCTTTACCCA 1201 GAAAAGTTCA ACAACAAAAC AAACGGTATCACTTTCCGTC GTTGGCTTAT 1251 GCATGCTAAC CCAAGATTGT CTCACTACTT GGATGAGATTCTTGGAGATG 1301 GTTGGCACCA TGAAGCAGAT GAGCTTGAAA AACTTTTGTC TTATGAAGAC1351 AAAGCAGCTG TCAAAGAAAA ATTGGAAAGC ATCAAGGCTC ACAACAAACG 1401TAAATTGGCT CGTCACTTGA AAGAACACCA AGGTGTGGAA ATCAATCCAA 1451 ATTCTATCTTTGATATCCAA ATCAAACGTC TTCACGAGTA CAAACGCCAA 1501 CAAATGAACG CTTTGTACGTGATCCACAAA TACCTTGACA TCAAAGCTGG 1551 TAACATCCCT GCTCGTCCAA TCACAATCTTCTTTGGTGGT AAAGCAGCTC 1601 CAGCCTACAC AATCGCTCAA GACATTATCC ATTTAATCCTTTGCATGTCA 1651 GAAGTTATTG CTAACGATCC AGCAGTAGCT CCACACTTGC AAGTAGTTAT1701 GGTTGAAAAC TACAACGTTA CTGCAGCAAG TTTCCTTATC CCAGCATGTG 1751ATATCTCAGA ACAAATCTCA CTTGCTTCTA AAGAAGCTTC AGGTACTGGT 1801 AACATGAAATTCATGTTGAA CGGAGCTTTG ACACTTGGTA CTATGGACGG 1851 TGCTAACGTG GAAATCGCTGAGTTGGTTGG AGAAGAAAAC ATCTACATCT 1901 TCGGTGAAGA TTCAGAAACT GTTATCGACCTTTACGCAAA AGCAGCTTAC 1951 AAATCAAGCG AATTCTACGC TCGTGAAGCT ATCAAACCATTGGTTGACTT 2001 CATCGTTAGT GATGCAGTTC TTGCAGCTGG AAACAAAGAG CGCTTGGAAC2051 GTCTTTACAA TGAATTGATC AACAAAGACT GGTTCATGAC TCTTCTTGAC 2101TTGGAAGACT ACATCAAAGT CAAAGAGCAA ATGCTTGCTG ACTACGAAGA 2151 CCGTGACGCATGGTTGGATA AAGTCATCGT TAACATTTCT AAAGCAGGAT 2201 TCTTCTCATC TGACCGTACAATCGCTCAGT ATAACGAAGA CATCTGGCAC 2251 TTGAACTAA -(R₂)_(n)-Y (D)Polypeptide sequence embodiments [SEQ ID NO:2]. X-(R₁)_(n)-1 MLSLQEFVQNRYNKTIAECS NEELYLALLN YSKLASSQKP VNTGKKKVYY 51 ISAEFLIGKL LSNNLINLGLYDDVKKELAA AGKDLIEVEE VELEPSLGNG 101 GLGRLAACFI DSIATLGLNG DGVGLNYHFGLFQQVLKNNQ QETIPNAWLT 151 EQNWLVRSSR SYQVPFADFT LTSTLYDIDV TGYETATKNRLRLFDLDSVD 201 SSIIKDGINF DKTDIARNLT LFLYPDDSDR QGELLRIFQQ YFMVSNGAQL251 IIDEAIEKGS NLHDLADYAV VQINDTHPSM VIPELIRLLT ARGIELDEAI 301SIVRSMTAYT NHTILAEALE KWPLEFLQEV VPHLVPIIEE LDRRVKAEYK 351 DPAVQIIDESGRVHMAHMDI HYGYSVNGVA ALHTEILKNS ELKAFYDLYP 401 EKFNNKTNGI TFRRWLMHHNPRLSHYLDEI LGDGWHHEAD ELEKLLSYED 451 KAAVKEKLES IKAANKRKLA RHLKEHQGVEINPNSIFDIQ IKRLHEYKRQ 501 QMNALYVIHK YLDIKAGNIP ARPITIFFGG KAAPAYTIAQDIIHLILCMS 551 EVIANDPAVA PHLQVVMVEN YNVTAASFLI PACDISEQIS LASKEASGTG601 NMKFMLNGAL TLGTMDGANV EIAELVGEEN IYIFGEDSET VIDLYAKAAY 651KSSEFYAREA IKPLVDFIVS DAVLAAGNKE RLERLYNELI NKDWFMTLLD 701 LEDYIKVKEQMLADYEDRDA WLDKVIVNIS KAGFFSSDRT IAQYNEDIWH 751 LN -(R₂)_(n)-y (D)Sequences from Streptococcus pneumoniae glycogen phosphorylasepolynucleotide ORF sequence [SEQ ID NO:3]. 5′- ATGTTATCAC TACAAGAATTTGTACAAAAT CGTTACAATA AAACCATTGC 51 AGAATGTAGC AATGAAGAGC TTTACCTTGCTCTTCTTAAC TACAGCAAGC 101 TTGCAAGCAG CCAAAAACCA GTCAACACTG GTAAGAAAAAAGTTTACTAC 151 ATCTCAGCTG AGTTCTTGAT TGGTAAACTC TTGTCAAACA ACTTGATTAA201 CCTTGGTCTT TACGACGATG TTAAAAAAGA ACTTGCAGCT GCAGGTAAAG 251ACTTGATCGA AGTTGAAGAA GTTGAATTGG AACCATCTCT TGGTAATGGT 301 GGTTTGGGACGTTTGGCTGC CTGCTTTATC GACTCAATTG CTACTCTTGG 351 TTTGAATGGT GACGGTGTTGGTCTTAACTA CCACTTTGGT CTTTTCCAAC 401 AAGTTCTTAA AAACAACCAA CAAGAAACAATTCCAAATGC ATGGTTGACA 451 GAGCAAAACT GGTTGGTTCG CTCAAGCCGT AGCTACCAAGTACCATTTGC 501 AGACTTTACT TTGACATCAA CTCTTTACGA TATTGATGTT ACTGGTTATG551 AAACAGCGAC TAAAAACCGC TTGCGTTTGT TTGACTTGGA TTCAGTTGAT 601TCTTCTATTA TTAAAGATGG TATCAACTTT GACAAGACAG ATATCGCTCG 651 CAACTTGACTCTCTTCCTTT ACCCAGATGA TAGTGACCGT CAAGGTGAAT 701 TGCTCCGTAT CTTCCAACAATACTTCATGG TTTCAAACGG TGCGCAATTG 751 ATCATCGACG AAGCAATCGA AAAAGGAAGCAACTTGCATG ACCTTGCTGA 1651 GAAGTTATTG CTAACGATCC AGCAGTAGCT CCACACTTGCAAGTAGTTAT 1701 GGTTGAAAAC TACAACGTTA CTGCAGCAAG TTTCCTTATC CCAGCATGTG1751 ATATCTCAGA ACAAATCTCA CTTGCTTCTA AAGAAGCTTC AGGTACTGGT 1801AACATGAAAT TCATGTTGAA CGGAGCTTTG ACACTTGGTA CTATGGACGG 1851 TGCTAACGTGGAAATCGCTG AGTTGGTTGG AGAAGAAAAC ATCTACATCT 1901 TCGGTGAAGA TTCAGAAACTGTTATCGACC TTTACGCAAA AGCAGCTTAC 1951 AAATCAAGCG AATTCTACGC TCGTGAAGCTATCAAACCAT TGGTTGACTT 2001 CATCGTTAGT GATGCAGTTC TTGCAGCTGG AAACAAAGAGCGCTTGGAAC 2051 GTCTTTACAA TGAATTGATC AACAAAGACT GGTTCATGAC TCTTCTTGAC2101 TTGGAAGACT ACATCAAAGT CAAAGAGCAA ATGCTTGCTG ACTACGAAGA 2151CCGTGACGCA TGGTTGGATA AAGTCATCGT TAACATTTCT AAAGCAGGAT 2201 TCTTCTCATCTGACCGTACA ATCGCTCAGT ATAACGAAGA CATCTGGCAC 2251 TTGAAC-3′ (F) glycogenphosphorylase polypeptide sequence deduced from the polynucleotide ORFsequence in this table [SEQ ID NO:4]. NH₂-1 MLSLQEFVQN RYNKTIAECSNEELYLALLN YSKLAS SQKP VNTGKKKVYY 51 ISAEFLIGKL LSNNLINLGL YDDVKKELAAAGKDLIEVEE VELEPSLGNG 101 GLGRLAACFI DSIATLGLNG DGVGLNYHFG LFQQVLKNNQQETIPNAWLT 151 EQNWLVRSSR SYQVPFADFT LTSTLYDIDV TGYETATKNR LRLFDLDSVD201 SSIIKDGINF DKTDIARNLT LFLYPDDSDR QGELLRIFQQ YFMVSNGAQL 251IIDEAIEKGS NLHDLADYAV VQINDTHPSM VIPELIRLLT ARGIELDEAI 301 SIVRSMTAYTNHTILAEALE KWPLEFLQEV VPHLVPIIEE LDRRVKAEYK 351 DPAVQIIDES GRVHMAHMDIHYGYSVNGVA ALHTEILKNS ELKAFYDLYP 401 EKFNNKTNGI TFRRWLMHAN PRLSHYLDEILGDGWHHEAD ELEKLLSYED 451 KAAVKEKLES IKAHNKRKLA RHLKEHQGVE INPNSIFDIQIKRLHEYKRQ 501 QMNALYVIHK YLDIKAGNIP ARPITIFFGG KAAPAYTIAQ DIIHLILCMS551 EVIANDPAVA PHLQVVMVEN YNVTAASFLI PACDISEQIS LASKEASGTG 601NMKFMLNGAL TLGTMDGANV EIAELVGEEN IYIFGEDSET VIDLYAAAAY 651 KSSEFYAREAIKPLVDFIVS DAVAAAGNKE RLERLYNELI NKDWFMTLLD 701 LEDYIKVKEQ MLADYEDRDAWLDKVIVNIS KAGFFSSDRT IAQYNEDIWH 751 LN-COOH

The invention further provides these primers with 1, 2, 3 or 4nucleotides removed from the 5′ and/or the 3′ end. These primers may beused for, among other things, amplifying glycogen phosphorylase DNAisolated from a sample derived from an individual. The primers may beused to amplify the gene isolated from an infected individual such thatthe gene may then be subject to various techniques for elucidation ofthe DNA sequence. In this way, mutations in the DNA sequence may bedetected and used to diagnose infection and to serotype and/or classifythe infectious agent.

The invention farther provides a process for diagnosing, disease,preferably bacterial infections, more preferably infections byStreptococcus pneumoniae, and most preferably otitis media,conjunctivitis, pneumonia, bacteremia, meningitis, sinusitis, pleuralempyema and endocarditis, and most particularly meningitis, such as forexample infection of cerebrospinal fluid, comprising determining from asample derived from an individual a increased level of expression ofpolynucleotide having the sequence of Table 1 [SEQ ID NO: 1]. Increasedor decreased expression of glycogen phosphorylase polynucleotide can bemeasured using any on of the methods well known in the art for thequantation of polynucleotides, such as, for example, amplification, PCR,RT-PCR, RNase protection, Northern blotting and other hybridizationmethods.

In addition, a diagnostic assay in accordance with the invention fordetecting over-expression of glycogen phosphorylase protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a glycogen phosphorylase protein, in a sample derived from ahost are well-known to those of skill in the art Such assay methodsinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis and ELISA assays.

Antibodies

The polypeptides of the invention or variants thereof, or cellsexpressing them can be used as an immunogen to produce antibodiesimmunospecific for such polypeptides. “Antibodies” as used hereinincludes monoclonal and polyclonal antibodies, chimeric, single chain,simianized antibodies and humanized antibodies, as well as Fabfragments, including the products of an Fab immunolglobulin expressionlibrary.

Antibodies generated against the polypeptides of the invention can beobtained by administering the polypeptides or epitope-bearing fragments,analogues or cells to an animal, preferably a nonhuman, using routineprotocols. For preparation of monoclonal antibodies, any technique knownin the art that provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etal., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

Techniques for the production of single chain antibodies (U.S. Pat. No.4,946,778) can be adapted to produce single chain antibodies topolypeptides of this invention. Also, transgenic mice, or otherorganisms such as other mammals, may be used to express humanizedantibodies.

Alternatively phage display technology may be utilized to selectantibody genes with binding activities towards the polypeptide eitherfrom repertoires of PCR amplified v-genes of lymphocytes from humansscreened for possessing anti-glycogen phosphorylase or from naivelibraries (McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J.et al., (1992) Biotechnology 10, 779-783). The affinity of theseantibodies can also be improved by chain shuffling (Clackson, T. et al.,(1991) Nature 352, 624-628).

If two antigen binding domains are present each domain may be directedagainst a different epitope—termed ‘bispecific’ antibodies.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptides to purify the polypeptides byaffinity chromatography.

Thus, among others, antibodies against glycogen phosphorylase-polypeptide may be employed to treat infections, particularly bacterialinfections and especially otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Polypeptide variants include antigenically, epitopically orimmunologically equivalent variants that form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognized by certain antibodies which, when raised to theprotein or polypeptide according to the invention, interfere with theimmediate physical interaction between pathogen and mammalian host. Theterm “immunologically equivalent derivative” as used herein encompassesa peptide or its equivalent which when used in a suitable formulation toraise antibodies in a vertebrate, the antibodies act to interfere withthe immediate physical interaction between pathogen and mammalian host.

The polypeptide, such as an antigenically or immunologically equivalentderivative or a fusion protein thereof is used as an antigen to immunizea mouse or other animal such as a rat or chicken. The fusion protein mayprovide stability to the polypeptide. The antigen may be associated, forexample by conjugation, with an immunogenic carrier protein for examplebovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

Preferably, the antibody or variant thereof is modified to make it lessimmunogenic in the individual. For example, if the individual is humanthe antibody may most preferably be “humanized” ; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal., (1991) Biotechnology 9, 266-273.

The use of a polynucleotide of the invention in genetic immunizationwill preferably employ a suitable delivery method such as directinjection of plasmid DNA into muscles (Wolff et al., Hum Mol Genet 1992,1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419), delivery of DNAcomplexed with specific protein carriers (Wu et al., J Biol Chem. 1989:264,16985), coprecipitation of DNA with calcium phosphate (Benvenisty &Reshef, PNAS USA, 1986:83,9551), encapsulation of DNA in various formsof liposomes (Kaneda et al., Science 1989:243,375), particle bombardment(rang et al., Nature 1992, 356:152, Eisenbraun et al., DNA Cell Biol1993, 12:791) and in vivo infection using cloned retroviral vectors(Seeger t al., PNAS USA 1984:81,5849).

Antagonists and Agonists—Assays and Molecules

Polypeptides of the invention may also be used to assess the binding ofsmall molecule substrates and ligands in, for example, cells, cell-freepreparations, chemical libraries, and natural product mixtures. Thesesubstrates and ligands may be natural substrates and ligands or may bestructural or functional mimetics. See, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5 (1991).

The invention also provides a method of screening compounds to identifythose which enhance (agonist) or block (antagonist) the action ofglycogen phosphorylase polypeptides or polynucleotides, particularlythose compounds that are bacteriostatic and/or bactericidal. The methodof screening may involve high-throughput techniques. For example, toscreen for antagonists or antagonists, a synthetic reaction mix, acellular compartment, such as a membrane, cell envelope or cell wall, ora preparation of any thereof, comprising glycogen phosphorylasepolypeptide and a labeled substrate or ligand of such polypeptide isincubated in the absence or the presence of a candidate molecule thatmay be a glycogen phosphorylase agonist or antagonist. The ability ofthe candidate molecule to agonize or antagonize the glycogenphosphorylase polypeptide is reflected in decreased binding of thelabeled ligand or decreased production of product from such substrate.Molecules that bind gratuitously, i.e., without inducing the effects ofglycogen phosphorylase polypeptide are most likely to be goodantagonists. Molecules that bind well and increase the rate of productproduction from substrate are agonists. Detection of the rate or levelof production of product from substrate may be enhanced by using areporter system. Reporter systems that may be useful in this regardinclude but are not limited to colorimetric labeled substrate convertedinto product, a reporter gene that is responsive to changes in glycogenphosphorylase polynucleotide or polypeptide activity, and binding assaysknown in the art

Another example of an assay for glycogen phosphorylase antagonists is acompetitive assay that combines glycogen phosphorylase and a potentialantagonist with glycogen phosphorylase-binding molecules, recombinantglycogen phosphorylase binding molecules, natural substrates or ligands,or substrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. Glycogen phosphorylase can be labeled,such as by radioactivity or a colorimetric compound, such that thenumber of glycogen phosphorylase molecules bound to a binding moleculeor converted to product can be determined accurately to assess theeffectiveness of the potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, a peptide, apolypeptide such as a closely related protein or antibody that binds thesame sites on a binding molecule, such as a binding molecule, withoutinducing glycogen phosphorylase-induced activities, thereby preventingthe action of glycogen phosphorylase by excluding glycogen phosphorylasefrom binding.

Potential antagonists include a small molecule that binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules. Otherpotential antagonists include antisense molecules (see Okano, J.Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSE INHIBITORSOF GENE EXPRESSION, CRC Press, Boca Raton, Fla. (1988), for adescription of these molecules). Preferred potential antagonists includecompounds related to and variants of glycogen phosphorylase.

Each of the DNA sequences provided herein may be used in the discoveryand development of antibacterial compounds. The encoded protein, uponexpression, can be used as a target for the screening of antibacterialdrugs. Additionally, the DNA sequences encoding the amino terminalregions of the encoded protein or Shine-Delgarno or other translationfacilitating sequences of the respective mRNA can be used to constructantisense sequences to control the expression of the coding sequence ofinterest.

The invention also provides the use of the polypeptide, polynucleotideor inhibitor of the invention to interfere with the initial physicalinteraction between a pathogen and mammalian host responsible forsequelae of infection. In particular the molecules of the invention maybe used: in the prevention of adhesion of bacteria, in particular grampositive bacteria, to mammalian extracellular matrix proteins onin-dwelling devices or to extracellular matrix proteins in wounds; toblock glycogen phosphorylase protein-mediated mammalian cell invasionby, for example, initiating phosphorylation of mammalian tyrosine kinase(Rosenshine et al., Infect. Immun. 60:2211 (1992); to block bacterialadhesion between mammalian extracellular matrix proteins and bacterialglycogen phosphorylase proteins that mediate tissue damage and; to blockthe normal progression of pathogenesis in infections initiated otherthan by the implantation of in-dwelling devices or by other surgicaltechniques.

The antagonists and agonists of the invention may be employed, forinstance, to inhibit and treat otitis media, conjunctivitis, pneumonia,bacteremia, meningitis, sinusitis, pleural empyema and endocarditis, andmost particularly meningitis, such as for example infection ofcerebrospinal fluid.

Vaccines

Another aspect of the invention relates to a method for inducing animmunological response in an individual, particularly a mammal whichcomprises inoculating the individual with glycogen phosphorylase, or afragment or variant thereof, adequate to produce antibody and/ or T cellimmune response to protect said individual from infection, particularlybacterial infection and most particularly Streptococcus pneumoniaeinfection. Also provided are methods whereby such immunological responseslows bacterial replication. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises delivering to such individual a nucleic acid vector to directexpression of glycogen phosphorylase, or a fragment or a variantthereof, for expressing glycogen phosphorylase, or a fragment or avariant thereof in vivo in order to induce an immunological response,such as, to produce antibody and/ or T cell immune response, including,for example, cytokine-producing T cells or cytotoxic T cells, to protectsaid individual from disease, whether that disease is alreadyestablished within the individual or not One way of administering thegene is by accelerating it into the desired cells as a coating onparticles or otherwise.

Such nucleic acid vector may comprise DNA, RNA, a modified nucleic acid,or a DNA/RNA hybrid.

A further aspect of the invention relates to an immunologicalcomposition which, when introduced into an individual capable or havinginduced within it an immunological response, induces an immunologicalresponse in such individual to a glycogen phosphorylase or protein codedtherefrom, wherein the composition comprises a recombinant glycogenphosphorylase or protein coded therefrom comprising DNA which codes forand expresses an antigen of said glycogen phosphorylase or protein codedtherefrom. The immunological response may be used therapeutically orprophylactically and may take the form of antibody immunity or cellularimmunity such as that arising from CTL or CD4+T cells.

A glycogen phosphorylase polypeptide or a fragment thereof may be fusedwith co-protein which may not by itself produce antibodies, but iscapable of stabilizing the first protein and producing a fused proteinwhich will have immunogenic and protective properties. Thus fusedrecombinant protein, preferably further comprises an antigenicco-protein, such as lipoprotein D from Hemophilus influenzae,Glutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilize the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

Provided by this invention are compositions, particularly vaccinecompositions, and methods comprising the polypeptides or polynucleotidesof the invention and immunostimulatory DNA sequences, such as thosedescribed in Sato, Y. et al. Science 273: 352 (1996).

Also, provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Streptococcus pneumoniae will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of bacterial infection, particularlyStreptococcus pneumoniae infection, in mammals, particularly humans.

The polypeptide may be used as an antigen for vaccination of a host toproduce specific antibodies which protect against invasion of bacteria,for example by blocking adherence of bacteria to damaged tissue.Examples of tissue damage include wounds in skin or connective tissuecaused, e.g., by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

The invention also includes a vaccine formulation which comprises animmunogenic recombinant protein of the invention together with asuitable carrier. Since the protein may be broken down in the stomach,it is preferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the bodily fluid, preferably the blood, of theindividual; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

While the invention has been described with reference to certainglycogen phosphorylase protein, it is to be understood that this coversfragments of the naturally occurring protein and similar proteins withadditions, deletions or substitutions which do not substantially affectthe immunogenic properties of the recombinant protein.

Compositions, Kits and Administration

The invention also relates to compositions comprising the polynucleotideor the polypeptides discussed above or their agonists or antagonists.The polypeptides of the invention may be employed in combination with anon-sterile or sterile carrier or carriers for use with cells, tissuesor organisms, such as a pharmaceutical carrier suitable foradministration to a subject Such compositions comprise, for instance, amedia additive or a therapeutically effective amount of a polypeptide ofthe invention and a pharmaceutically acceptable carrier or excipient.Such carriers may include, but are not limited to, saline, bufferedsaline, dextrose, water, glycerol, ethanol and combinations thereof Theformulation should suit the mode of administration. The inventionfurther relates to diagnostic and pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Polypeptides and other compounds of the invention may be employed aloneor in conjunction with other compounds, such as therapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes among others.

In therapy or as a prophylactic, the active agent may be administered toan individual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

Alternatively the composition may be formulated for topical applicationfor example in the form of ointments, creams, lotions, eye ointments,eye drops, ear drops, mouthwash, impregnated dressings and sutures andaerosols, and may contain appropriate conventional additives, including,for example, preservatives, solvents to assist drug penetration, andemollients in ointments and creams. Such topical formulations may alsocontain compatible conventional carriers, for example cream or ointmentbases, and ethanol or oleyl alcohol for lotions. Such carriers mayconstitute from about 1% to about 98% by weight of the formulation; moreusually they will constitute up to about 80% by weight of theformulation.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage level of the active agent will be from 0.01 mg/kgto 10 mg/kg, typically around 1 mg/kg. The physician in any event willdetermine the actual dosage which will be most suitable for anindividual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

Indwelling devices include surgical implants, prosthetic devices andcatheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters.

The composition of the invention may be administered by injection toachieve ii systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent bacterial wound infections, especiallyStreptococcus pneumoniae wound infections.

Many orthopaedic surgeons consider that humans with prosthetic jointsshould be considered for antibiotic prophylaxis before dental treatmentthat could produce a bacteremia. Late deep infection is a seriouscomplication sometimes leading to loss of the prosthetic joint and isaccompanied by significant morbidity and mortality. It may therefore bepossible to extend the use of the active agent as a replacement forprophylactic antibiotics in this situation.

In addition to the therapy described above, the compositions of thisinvention may be used generally as a wound treatment agent to preventadhesion of bacteria to matrix proteins exposed in wound tissue and forprophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

Alternatively, the composition of the invention may be used to bathe anindwelling device immediately before insertion. The active agent willpreferably be present at a concentration of 1 82 g/ml to 10 mg/ml forbathing of wounds or indwelling devices.

A vaccine composition is conveniently in injectable form. Conventionaladjuvants may be employed to enhance the immune response. A suitableunit dose for vaccination is 0.5-5 microgram/kg of antigen, and suchdose is preferably administered 1-3 times and with an interval of 1-3weeks. With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

Each reference disclosed herein is incorporated by reference herein inits entirety. Any patent application to which this application claimspriority is also incorporated by reference herein in its entirety.

EXAMPLES

The examples below are carried out using standard techniques, which arewell known and routine to those of skill in the art, except whereotherwise described in detail. The examples are illustrative, but do notlimit the invention.

Example 1 Strain Selection, Library Production and Sequencing

The polynucleotide having the DNA sequence given in SEQ ID NO:l wasobtained from a library of clones of chromosomal DNA of Streptococcuspneumoniae in E. coli. The sequencing data from two or more clonescontaining overlapping Streptococcus pneumoniae DNAs was used toconstruct the contiguous DNA sequence in SEQ ID NO: 1. Libraries may beprepared by routine methods, for example:

Methods 1 and 2 Below

Total cellular DNA is isolated from Streptococcus pneumoniae 0100993according to standard procedures and size-fractionated by either of twomethods.

Method 1

Total cellular DNA is mechanically sheared by passage through a needlein order to size-fractionate according to standard procedures. DNAfragments of up to 11 kbp in size are rendered blunt by treatment withexonuclease and DNA polymerase, and EcoRI linkers added. Fragments areligated into the vector Lambda ZapII that has been cut with EcoRI, thelibrary packaged by standard procedures and E. coli infected with thepackaged library. The library is amplified by standard procedures.

Method 2

Total cellular DNA is partially hydrolyzed with a one or a combinationof restriction enzymes appropriate to generate a series of fragments forcloning into library vectors (e.g., RsaI, PalI, AluI, Bshl235I), andsuch fragments are size-fractionated according to standard procedures.EcoRI linkers are ligated to the DNA and the fragments then ligated intothe vector Lambda ZapII that have been cut with EcoRI, the librarypackaged by standard procedures, and E. coli infected with the packagedlibrary. The library is amplified by standard procedures.

6 2259 base pairs nucleic acid double linear unknown 1 ATGTTATCACTACAAGAATT TGTACAAAAT CGTTACAATA AAACCATTGC AGAATGTAGC 60 AATGAAGAGCTTTACCTTGC TCTTCTTAAC TACAGCAAGC TTGCAAGCAG CCAAAAACCA 120 GTCAACACTGGTAAGAAAAA AGTTTACTAC ATCTCAGCTG AGTTCTTGAT TGGTAAACTC 180 TTGTCAAACAACTTGATTAA CCTTGGTCTT TACGACGATG TTAAAAAAGA ACTTGCAGCT 240 GCAGGTAAAGACTTGATCGA AGTTGAAGAA GTTGAATTGG AACCATCTCT TGGTAATGGT 300 GGTTTGGGACGTTTGGCTGC CTGCTTTATC GACTCAATTG CTACTCTTGG TTTGAATGGT 360 GACGGTGTTGGTCTTAACTA CCACTTTGGT CTTTTCCAAC AAGTTCTTAA AAACAACCAA 420 CAAGAAACAATTCCAAATGC ATGGTTGACA GAGCAAAACT GGTTGGTTCG CTCAAGCCGT 480 AGCTACCAAGTACCATTTGC AGACTTTACT TTGACATCAA CTCTTTACGA TATTGATGTT 540 ACTGGTTATGAAACAGCGAC TAAAAACCGC TTGCGTTTGT TTGACTTGGA TTCAGTTGAT 600 TCTTCTATTATTAAAGATGG TATCAACTTT GACAAGACAG ATATCGCTCG CAACTTGACT 660 CTCTTCCTTTACCCAGATGA TAGTGACCGT CAAGGTGAAT TGCTCCGTAT CTTCCAACAA 720 TACTTCATGGTTTCAAACGG TGCGCAATTG ATCATCGACG AAGCAATCGA AAAAGGAAGC 780 AACTTGCATGACCTTGCTGA CTACGCAGTT GTCCAAATCA ACGATACTCA CCCATCAATG 840 GTGATTCCTGAATTGATTCG TCTTTTGACT GCACGTGGTA TCGAGCTTGA CGAAGCAATC 900 TCAATTGTTCGTAGCATGAC TGCCTACACT AACCACACAA TCCTTGCTGA GGCGCTTGAA 960 AAATGGCCTCTTGAATTCTT GCAAGAAGTG GTTCCTCACT TGGTACCAAT CATCGAAGAA 1020 TTGGACCGTCGTGTGAAGGC AGAGTACAAA GATCCAGCTG TTCAAATCAT CGATGAGAGC 1080 GGACGTGTTCACATGGCTCA CATGGATATC CACTACGGAT ACAGTGTTAA CGGGGTTGCA 1140 GCACTTCATACTGAAATCTT GAAAAATTCT GAGTTGAAAG CCTTCTACGA CCTTTACCCA 1200 GAAAAGTTCAACAACAAAAC AAACGGTATC ACTTTCCGTC GTTGGCTTAT GCATGCTAAC 1260 CCAAGATTGTCTCACTACTT GGATGAGATT CTTGGAGATG GTTGGCACCA TGAAGCAGAT 1320 GAGCTTGAAAAACTTTTGTC TTATGAAGAC AAAGCAGCTG TCAAAGAAAA ATTGGAAAGC 1380 ATCAAGGCTCACAACAAACG TAAATTGGCT CGTCACTTGA AAGAACACCA AGGTGTGGAA 1440 ATCAATCCAAATTCTATCTT TGATATCCAA ATCAAACGTC TTCACGAGTA CAAACGCCAA 1500 CAAATGAACGCTTTGTACGT GATCCACAAA TACCTTGACA TCAAAGCTGG TAACATCCCT 1560 GCTCGTCCAATCACAATCTT CTTTGGTGGT AAAGCAGCTC CAGCCTACAC AATCGCTCAA 1620 GACATTATCCATTTAATCCT TTGCATGTCA GAAGTTATTG CTAACGATCC AGCAGTAGCT 1680 CCACACTTGCAAGTAGTTAT GGTTGAAAAC TACAACGTTA CTGCAGCAAG TTTCCTTATC 1740 CCAGCATGTGATATCTCAGA ACAAATCTCA CTTGCTTCTA AAGAAGCTTC AGGTACTGGT 1800 AACATGAAATTCATGTTGAA CGGAGCTTTG ACACTTGGTA CTATGGACGG TGCTAACGTG 1860 GAAATCGCTGAGTTGGTTGG AGAAGAAAAC ATCTACATCT TCGGTGAAGA TTCAGAAACT 1920 GTTATCGACCTTTACGCAAA AGCAGCTTAC AAATCAAGCG AATTCTACGC TCGTGAAGCT 1980 ATCAAACCATTGGTTGACTT CATCGTTAGT GATGCAGTTC TTGCAGCTGG AAACAAAGAG 2040 CGCTTGGAACGTCTTTACAA TGAATTGATC AACAAAGACT GGTTCATGAC TCTTCTTGAC 2100 TTGGAAGACTACATCAAAGT CAAAGAGCAA ATGCTTGCTG ACTACGAAGA CCGTGACGCA 2160 TGGTTGGATAAAGTCATCGT TAACATTTCT AAAGCAGGAT TCTTCTCATC TGACCGTACA 2220 ATCGCTCAGTATAACGAAGA CATCTGGCAC TTGAACTAA 2259 752 amino acids amino acid singlelinear unknown 2 Met Leu Ser Leu Gln Glu Phe Val Gln Asn Arg Tyr Asn LysThr Ile 1 5 10 15 Ala Glu Cys Ser Asn Glu Glu Leu Tyr Leu Ala Leu LeuAsn Tyr Ser 20 25 30 Lys Leu Ala Ser Ser Gln Lys Pro Val Asn Thr Gly LysLys Lys Val 35 40 45 Tyr Tyr Ile Ser Ala Glu Phe Leu Ile Gly Lys Leu LeuSer Asn Asn 50 55 60 Leu Ile Asn Leu Gly Leu Tyr Asp Asp Val Lys Lys GluLeu Ala Ala 65 70 75 80 Ala Gly Lys Asp Leu Ile Glu Val Glu Glu Val GluLeu Glu Pro Ser 85 90 95 Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ala CysPhe Ile Asp Ser 100 105 110 Ile Ala Thr Leu Gly Leu Asn Gly Asp Gly ValGly Leu Asn Tyr His 115 120 125 Phe Gly Leu Phe Gln Gln Val Leu Lys AsnAsn Gln Gln Glu Thr Ile 130 135 140 Pro Asn Ala Trp Leu Thr Glu Gln AsnTrp Leu Val Arg Ser Ser Arg 145 150 155 160 Ser Tyr Gln Val Pro Phe AlaAsp Phe Thr Leu Thr Ser Thr Leu Tyr 165 170 175 Asp Ile Asp Val Thr GlyTyr Glu Thr Ala Thr Lys Asn Arg Leu Arg 180 185 190 Leu Phe Asp Leu AspSer Val Asp Ser Ser Ile Ile Lys Asp Gly Ile 195 200 205 Asn Phe Asp LysThr Asp Ile Ala Arg Asn Leu Thr Leu Phe Leu Tyr 210 215 220 Pro Asp AspSer Asp Arg Gln Gly Glu Leu Leu Arg Ile Phe Gln Gln 225 230 235 240 TyrPhe Met Val Ser Asn Gly Ala Gln Leu Ile Ile Asp Glu Ala Ile 245 250 255Glu Lys Gly Ser Asn Leu His Asp Leu Ala Asp Tyr Ala Val Val Gln 260 265270 Ile Asn Asp Thr His Pro Ser Met Val Ile Pro Glu Leu Ile Arg Leu 275280 285 Leu Thr Ala Arg Gly Ile Glu Leu Asp Glu Ala Ile Ser Ile Val Arg290 295 300 Ser Met Thr Ala Tyr Thr Asn His Thr Ile Leu Ala Glu Ala LeuGlu 305 310 315 320 Lys Trp Pro Leu Glu Phe Leu Gln Glu Val Val Pro HisLeu Val Pro 325 330 335 Ile Ile Glu Glu Leu Asp Arg Arg Val Lys Ala GluTyr Lys Asp Pro 340 345 350 Ala Val Gln Ile Ile Asp Glu Ser Gly Arg ValHis Met Ala His Met 355 360 365 Asp Ile His Tyr Gly Tyr Ser Val Asn GlyVal Ala Ala Leu His Thr 370 375 380 Glu Ile Leu Lys Asn Ser Glu Leu LysAla Phe Tyr Asp Leu Tyr Pro 385 390 395 400 Glu Lys Phe Asn Asn Lys ThrAsn Gly Ile Thr Phe Arg Arg Trp Leu 405 410 415 Met His Ala Asn Pro ArgLeu Ser His Tyr Leu Asp Glu Ile Leu Gly 420 425 430 Asp Gly Trp His HisGlu Ala Asp Glu Leu Glu Lys Leu Leu Ser Tyr 435 440 445 Glu Asp Lys AlaAla Val Lys Glu Lys Leu Glu Ser Ile Lys Ala His 450 455 460 Asn Lys ArgLys Leu Ala Arg His Leu Lys Glu His Gln Gly Val Glu 465 470 475 480 IleAsn Pro Asn Ser Ile Phe Asp Ile Gln Ile Lys Arg Leu His Glu 485 490 495Tyr Lys Arg Gln Gln Met Asn Ala Leu Tyr Val Ile His Lys Tyr Leu 500 505510 Asp Ile Lys Ala Gly Asn Ile Pro Ala Arg Pro Ile Thr Ile Phe Phe 515520 525 Gly Gly Lys Ala Ala Pro Ala Tyr Thr Ile Ala Gln Asp Ile Ile His530 535 540 Leu Ile Leu Cys Met Ser Glu Val Ile Ala Asn Asp Pro Ala ValAla 545 550 555 560 Pro His Leu Gln Val Val Met Val Glu Asn Tyr Asn ValThr Ala Ala 565 570 575 Ser Phe Leu Ile Pro Ala Cys Asp Ile Ser Glu GlnIle Ser Leu Ala 580 585 590 Ser Lys Glu Ala Ser Gly Thr Gly Asn Met LysPhe Met Leu Asn Gly 595 600 605 Ala Leu Thr Leu Gly Thr Met Asp Gly AlaAsn Val Glu Ile Ala Glu 610 615 620 Leu Val Gly Glu Glu Asn Ile Tyr IlePhe Gly Glu Asp Ser Glu Thr 625 630 635 640 Val Ile Asp Leu Tyr Ala LysAla Ala Tyr Lys Ser Ser Glu Phe Tyr 645 650 655 Ala Arg Glu Ala Ile LysPro Leu Val Asp Phe Ile Val Ser Asp Ala 660 665 670 Val Leu Ala Ala GlyAsn Lys Glu Arg Leu Glu Arg Leu Tyr Asn Glu 675 680 685 Leu Ile Asn LysAsp Trp Phe Met Thr Leu Leu Asp Leu Glu Asp Tyr 690 695 700 Ile Lys ValLys Glu Gln Met Leu Ala Asp Tyr Glu Asp Arg Asp Ala 705 710 715 720 TrpLeu Asp Lys Val Ile Val Asn Ile Ser Lys Ala Gly Phe Phe Ser 725 730 735Ser Asp Arg Thr Ile Ala Gln Tyr Asn Glu Asp Ile Trp His Leu Asn 740 745750 2256 base pairs nucleic acid double linear unknown 3 ATGTTATCACTACAAGAATT TGTACAAAAT CGTTACAATA AAACCATTGC AGAATGTAGC 60 AATGAAGAGCTTTACCTTGC TCTTCTTAAC TACAGCAAGC TTGCAAGCAG CCAAAAACCA 120 GTCAACACTGGTAAGAAAAA AGTTTACTAC ATCTCAGCTG AGTTCTTGAT TGGTAAACTC 180 TTGTCAAACAACTTGATTAA CCTTGGTCTT TACGACGATG TTAAAAAAGA ACTTGCAGCT 240 GCAGGTAAAGACTTGATCGA AGTTGAAGAA GTTGAATTGG AACCATCTCT TGGTAATGGT 300 GGTTTGGGACGTTTGGCTGC CTGCTTTATC GACTCAATTG CTACTCTTGG TTTGAATGGT 360 GACGGTGTTGGTCTTAACTA CCACTTTGGT CTTTTCCAAC AAGTTCTTAA AAACAACCAA 420 CAAGAAACAATTCCAAATGC ATGGTTGACA GAGCAAAACT GGTTGGTTCG CTCAAGCCGT 480 AGCTACCAAGTACCATTTGC AGACTTTACT TTGACATCAA CTCTTTACGA TATTGATGTT 540 ACTGGTTATGAAACAGCGAC TAAAAACCGC TTGCGTTTGT TTGACTTGGA TTCAGTTGAT 600 TCTTCTATTATTAAAGATGG TATCAACTTT GACAAGACAG ATATCGCTCG CAACTTGACT 660 CTCTTCCTTTACCCAGATGA TAGTGACCGT CAAGGTGAAT TGCTCCGTAT CTTCCAACAA 720 TACTTCATGGTTTCAAACGG TGCGCAATTG ATCATCGACG AAGCAATCGA AAAAGGAAGC 780 AACTTGCATGACCTTGCTGA CTACGCAGTT GTCCAAATCA ACGATACTCA CCCATCAATG 840 GTGATTCCTGAATTGATTCG TCTTTTGACT GCACGTGGTA TCGAGCTTGA CGAAGCAATC 900 TCAATTGTTCGTAGCATGAC TGCCTACACT AACCACACAA TCCTTGCTGA GGCGCTTGAA 960 AAATGGCCTCTTGAATTCTT GCAAGAAGTG GTTCCTCACT TGGTACCAAT CATCGAAGAA 1020 TTGGACCGTCGTGTGAAGGC AGAGTACAAA GATCCAGCTG TTCAAATCAT CGATGAGAGC 1080 GGACGTGTTCACATGGCTCA CATGGATATC CACTACGGAT ACAGTGTTAA CGGGGTTGCA 1140 GCACTTCATACTGAAATCTT GAAAAATTCT GAGTTGAAAG CCTTCTACGA CCTTTACCCA 1200 GAAAAGTTCAACAACAAAAC AAACGGTATC ACTTTCCGTC GTTGGCTTAT GCATGCTAAC 1260 CCAAGATTGTCTCACTACTT GGATGAGATT CTTGGAGATG GTTGGCACCA TGAAGCAGAT 1320 GAGCTTGAAAAACTTTTGTC TTATGAAGAC AAAGCAGCTG TCAAAGAAAA ATTGGAAAGC 1380 ATCAAGGCTCACAACAAACG TAAATTGGCT CGTCACTTGA AAGAACACCA AGGTGTGGAA 1440 ATCAATCCAAATTCTATCTT TGATATCCAA ATCAAACGTC TTCACGAGTA CAAACGCCAA 1500 CAAATGAACGCTTTGTACGT GATCCACAAA TACCTTGACA TCAAAGCTGG TAACATCCCT 1560 GCTCGTCCAATCACAATCTT CTTTGGTGGT AAAGCAGCTC CAGCCTACAC AATCGCTCAA 1620 GACATTATCCATTTAATCCT TTGCATGTCA GAAGTTATTG CTAACGATCC AGCAGTAGCT 1680 CCACACTTGCAAGTAGTTAT GGTTGAAAAC TACAACGTTA CTGCAGCAAG TTTCCTTATC 1740 CCAGCATGTGATATCTCAGA ACAAATCTCA CTTGCTTCTA AAGAAGCTTC AGGTACTGGT 1800 AACATGAAATTCATGTTGAA CGGAGCTTTG ACACTTGGTA CTATGGACGG TGCTAACGTG 1860 GAAATCGCTGAGTTGGTTGG AGAAGAAAAC ATCTACATCT TCGGTGAAGA TTCAGAAACT 1920 GTTATCGACCTTTACGCAAA AGCAGCTTAC AAATCAAGCG AATTCTACGC TCGTGAAGCT 1980 ATCAAACCATTGGTTGACTT CATCGTTAGT GATGCAGTTC TTGCAGCTGG AAACAAAGAG 2040 CGCTTGGAACGTCTTTACAA TGAATTGATC AACAAAGACT GGTTCATGAC TCTTCTTGAC 2100 TTGGAAGACTACATCAAAGT CAAAGAGCAA ATGCTTGCTG ACTACGAAGA CCGTGACGCA 2160 TGGTTGGATAAAGTCATCGT TAACATTTCT AAAGCAGGAT TCTTCTCATC TGACCGTACA 2220 ATCGCTCAGTATAACGAAGA CATCTGGCAC TTGAAC 2256 752 amino acids amino acid singlelinear unknown 4 Met Leu Ser Leu Gln Glu Phe Val Gln Asn Arg Tyr Asn LysThr Ile 1 5 10 15 Ala Glu Cys Ser Asn Glu Glu Leu Tyr Leu Ala Leu LeuAsn Tyr Ser 20 25 30 Lys Leu Ala Ser Ser Gln Lys Pro Val Asn Thr Gly LysLys Lys Val 35 40 45 Tyr Tyr Ile Ser Ala Glu Phe Leu Ile Gly Lys Leu LeuSer Asn Asn 50 55 60 Leu Ile Asn Leu Gly Leu Tyr Asp Asp Val Lys Lys GluLeu Ala Ala 65 70 75 80 Ala Gly Lys Asp Leu Ile Glu Val Glu Glu Val GluLeu Glu Pro Ser 85 90 95 Leu Gly Asn Gly Gly Leu Gly Arg Leu Ala Ala CysPhe Ile Asp Ser 100 105 110 Ile Ala Thr Leu Gly Leu Asn Gly Asp Gly ValGly Leu Asn Tyr His 115 120 125 Phe Gly Leu Phe Gln Gln Val Leu Lys AsnAsn Gln Gln Glu Thr Ile 130 135 140 Pro Asn Ala Trp Leu Thr Glu Gln AsnTrp Leu Val Arg Ser Ser Arg 145 150 155 160 Ser Tyr Gln Val Pro Phe AlaAsp Phe Thr Leu Thr Ser Thr Leu Tyr 165 170 175 Asp Ile Asp Val Thr GlyTyr Glu Thr Ala Thr Lys Asn Arg Leu Arg 180 185 190 Leu Phe Asp Leu AspSer Val Asp Ser Ser Ile Ile Lys Asp Gly Ile 195 200 205 Asn Phe Asp LysThr Asp Ile Ala Arg Asn Leu Thr Leu Phe Leu Tyr 210 215 220 Pro Asp AspSer Asp Arg Gln Gly Glu Leu Leu Arg Ile Phe Gln Gln 225 230 235 240 TyrPhe Met Val Ser Asn Gly Ala Gln Leu Ile Ile Asp Glu Ala Ile 245 250 255Glu Lys Gly Ser Asn Leu His Asp Leu Ala Asp Tyr Ala Val Val Gln 260 265270 Ile Asn Asp Thr His Pro Ser Met Val Ile Pro Glu Leu Ile Arg Leu 275280 285 Leu Thr Ala Arg Gly Ile Glu Leu Asp Glu Ala Ile Ser Ile Val Arg290 295 300 Ser Met Thr Ala Tyr Thr Asn His Thr Ile Leu Ala Glu Ala LeuGlu 305 310 315 320 Lys Trp Pro Leu Glu Phe Leu Gln Glu Val Val Pro HisLeu Val Pro 325 330 335 Ile Ile Glu Glu Leu Asp Arg Arg Val Lys Ala GluTyr Lys Asp Pro 340 345 350 Ala Val Gln Ile Ile Asp Glu Ser Gly Arg ValHis Met Ala His Met 355 360 365 Asp Ile His Tyr Gly Tyr Ser Val Asn GlyVal Ala Ala Leu His Thr 370 375 380 Glu Ile Leu Lys Asn Ser Glu Leu LysAla Phe Tyr Asp Leu Tyr Pro 385 390 395 400 Glu Lys Phe Asn Asn Lys ThrAsn Gly Ile Thr Phe Arg Arg Trp Leu 405 410 415 Met His Ala Asn Pro ArgLeu Ser His Tyr Leu Asp Glu Ile Leu Gly 420 425 430 Asp Gly Trp His HisGlu Ala Asp Glu Leu Glu Lys Leu Leu Ser Tyr 435 440 445 Glu Asp Lys AlaAla Val Lys Glu Lys Leu Glu Ser Ile Lys Ala His 450 455 460 Asn Lys ArgLys Leu Ala Arg His Leu Lys Glu His Gln Gly Val Glu 465 470 475 480 IleAsn Pro Asn Ser Ile Phe Asp Ile Gln Ile Lys Arg Leu His Glu 485 490 495Tyr Lys Arg Gln Gln Met Asn Ala Leu Tyr Val Ile His Lys Tyr Leu 500 505510 Asp Ile Lys Ala Gly Asn Ile Pro Ala Arg Pro Ile Thr Ile Phe Phe 515520 525 Gly Gly Lys Ala Ala Pro Ala Tyr Thr Ile Ala Gln Asp Ile Ile His530 535 540 Leu Ile Leu Cys Met Ser Glu Val Ile Ala Asn Asp Pro Ala ValAla 545 550 555 560 Pro His Leu Gln Val Val Met Val Glu Asn Tyr Asn ValThr Ala Ala 565 570 575 Ser Phe Leu Ile Pro Ala Cys Asp Ile Ser Glu GlnIle Ser Leu Ala 580 585 590 Ser Lys Glu Ala Ser Gly Thr Gly Asn Met LysPhe Met Leu Asn Gly 595 600 605 Ala Leu Thr Leu Gly Thr Met Asp Gly AlaAsn Val Glu Ile Ala Glu 610 615 620 Leu Val Gly Glu Glu Asn Ile Tyr IlePhe Gly Glu Asp Ser Glu Thr 625 630 635 640 Val Ile Asp Leu Tyr Ala LysAla Ala Tyr Lys Ser Ser Glu Phe Tyr 645 650 655 Ala Arg Glu Ala Ile LysPro Leu Val Asp Phe Ile Val Ser Asp Ala 660 665 670 Val Leu Ala Ala GlyAsn Lys Glu Arg Leu Glu Arg Leu Tyr Asn Glu 675 680 685 Leu Ile Asn LysAsp Trp Phe Met Thr Leu Leu Asp Leu Glu Asp Tyr 690 695 700 Ile Lys ValLys Glu Gln Met Leu Ala Asp Tyr Glu Asp Arg Asp Ala 705 710 715 720 TrpLeu Asp Lys Val Ile Val Asn Ile Ser Lys Ala Gly Phe Phe Ser 725 730 735Ser Asp Arg Thr Ile Ala Gln Tyr Asn Glu Asp Ile Trp His Leu Asn 740 745750 23 base pairs nucleic acid single linear unknown 5 GTACAAAATCGTTACAATAA AAC 23 22 base pairs nucleic acid single linear unknown 6CATCTTTAAT AATAGAAGAA TC 22

What is claimed is:
 1. An isolated polypeptide comprising a heterologousamino acid sequence fused to SEQ ID NO:2.
 2. A composition comprisingthe isolated polypeptide of claim 1 and a pharmaceutically acceptablecarrier.